1. Field of the Invention
This invention pertains to the general field of vascular and heart surgeries. In particular, it provides autologous and synthetic vascular and heart implants that possess an internal monolayer of endothelial cells genetically modified to express at least one of a number of therapeutic agents useful for the inhibition of intimal thickening.
2. Description of the Prior Art
Angioplasty and reconstructive vascular surgery are routinely-utilized surgical procedures for the treatment of arteriosclerosis, such as atherosclerosis and medial arteriosclerosis, heart and renal failure, arterial aneurysms, and other conditions that require general vascular bypass to restore blood flow to areas of ischemia. These techniques normally involve injury to a portion of an artery or vein followed by implantation of a donor or synthetic vascular graft, stent, or other implant in order to replace or repair the injured vascular or heart portion. The term "graft" is well understood by those skilled in the art and refers to unattached tissue or material, whether synthetic or naturally occurring, that is implanted, or intended to be implanted, into the body. Additionally, the term "stent" is well understood by those skilled in the art and refers to a prosthesis lying or intended to lie within tubular structures in the body in order to provide support to that tubular structure. As utilized herein, the term "implant" is meant to include, but not to be limited to, all intravascular devices, whether autologous or synthetic, vascular prostheses, artificial hearts, heart valves, vascular stents, and vascular grafts. As utilized herein, the term "vascular" is meant to refer collectively to all tissues, or synthetic materials replacing or intending to replace such tissues, that directly contact flowing blood from veins and arteries and that possess an endothelium, such as, for example, blood vessels and heart tissues. Though the vascular grafting techniques discussed above are commonly utilized, they possess an alarmingly high rate of complication (between 30-50%).
There exist two principal causes of vascular graft failure: thrombosis and smooth muscle cell proliferation.
Thrombosis
The first principal cause of vascular graft failure is the development of blood clots (thrombosis) at the site of vascular injury, which constrict or close the arterial passageway, known in the art as the vascular lumen, and lead to decreased blood flow (ischemia) to tissues and organs. The reaction that leads to the clotting of certain blood cells, which are known as platelets, is catalyzed by a protein complex named thrombin, which forms in the blood in response to various stimuli. Thrombin is a multifunctional protease that induces platelet aggregation and stimulation and the activation of coagulation-stimulating factors, both of which lead to thrombosis. Paradoxically, thrombin also possesses antithrombotic properties, which depend almost entirely upon interactions with healthy endothelial cells. Endothelial cells are specialized cells that form the innermost cellular wall of veins and arteries, which is referred to in the art as the endothelium, as well as forming the inner lining of the heart. The normal role of healthy endothelial cells is to provide a thromboresistant and actively anti-thrombogenic surface that inhibits the formation of clots and the prothrombotic function of thrombin and that does not allow platelets or other blood cells to adhere to the walls of the endothelium. Endothelial cells accomplish this antithrombotic role in part by the synthesis of physiological factors, such as prostacyclin, nitric oxide, ecto-adenosine diphosphatase, tissue-plasminogen activator inhibitor-1, thrombo-modulin, protein S, and heparan sulfate proteoglycan. The antithrombotic action of several of these factors are either thrombin-mediated or thrombin-activated, and thus a substantial portion of the anti-thrombogenic function of endotheilial cells requires direct interaction with thrombin. Additionally, endothelial cells also block the actions of pro-coagulant and prothrombotic molecules produced in the subendothelial matrix. Some additional biological factors produced by or interacting with endothelial cells that affect thrombogenecity include: plasminogen activator, soluble CD-4, Factor VIII, Factor IX, von Wildebrand Factor, urokinase, interferons, tumor necrosis factor, interleukins, hematopoietic growth factor, antibodies, glucocerebrosidase, ADA, phenylalanine, hydroxylase, human growth hormone, insulin, and erythropoietin. As utilized herein, the term "therapeutic agents" is meant to refer collectively to all of the above-listed physiological agents and biological factors. Thus, endothelial cells provide a vast array of mechanisms that inhibit thrombus formation, and the loss of endothelial cell function, such as that loss resulting from cellular damage caused by vascular surgery, causes a marked shift in the homeostatic balance toward thrombosis. Indeed, all angioplastic surgeries cause removal of or significant damage to the endothelial cell lining of the target blood vessel and, thereby, this surgical procedure results in a blood contacting surface that is extremely thrombogenic.
To overcome this first principal cause of vascular graft failure, researchers have attempted to create vascular grafts with a surface that is thromboresistant, with the majority of these efforts directed toward an improved polymer surface. Perhaps the ideal blood-surface interface is the naturally occurring human endothelium. If present on a prosthetic graft, it would offer many of the advantages of a native vessel. Unfortunately, endothelialization occurs spontaneously only to a limited degree in prosthetic grafts when placed into humans.
Seeding endothelial cells onto preclotted prosthetic grafts prior to implantation has accelerated the formation of an endothelial cell coverage of grafts in animals, but this technique has had limited use in humans. For a thorough listing of such experiments, see U.S. Pat. No. 5,131,907, entitled "Method of Treating a synthetic Naturally Occurring Surface with a Collagen Laminate to Support Microvascular Endothelial Cell Growth, and the Surface Itself," which is incorporated by reference herein in its entirety.
Endothelial cells from animal sources have been studied in culture since the 1920's. In 1973, Jaffe et al. successfully cultured endothelial cells from human umbilical veins, and these cells have been characterized functionally. See Jaffe et al., "Synthesis of Antihemophilia Factor Antigen by Cultured Human Endothelial Cells", J Clin Invest 1973;55:2757-64; Lewis, "Endothelium in Tissue Culture", Am J Anat 1922;30:39-59; and Jaffe et al., "Culture of Human Endothelial Cells Derived From Umbilical Veins", J Clin Invest 1973;52:2745-56. These cell cultures demonstrate a very limited growth potential, but the total number of cells produced from a single umbilical vein is usually quite limited, in the range of a 10-100-fold increase in harvested endothelial cells.
While several techniques have been proposed to increase the number of cells produced by the use of human umbilical vein endothelial cells, the ability to culture endothelial cells in large number remains less than ideal. Investigators have had limited success in culturing human and adult endothelial cells from pulmonary arteries and veins, but only for short periods of time. It has also been shown that human iliac artery endothelial cells may be cultured for a short number of passages. In a study by Glasberg et al., for example, it is reported that 50 to 500 viable cells can be obtained per 5-inch vessel segment, a very low yield. "Cultured Endothelial Cells Derived From Human Iliac Arteries", In Vitro 1982;18:859-66. Fry et al. have also reported successfully culturing human adult endothelial cells from abdominal arteries removed at the time of cadaver donor nephrectomy, but these cells also demonstrated limited proliferative capacity.
It is apparent from existing techniques that it is difficult to produce enough cells to preendothelialize a graft with a reasonable amount of vessel from the donor patient. Rather than completely endothelializing a graft prior to implantation, the concept of subconfluent "seeding" of a preclotted graft developed. Seeding vascular grafts with autogenous endothelial cells has recently been shown to increase the rate of endothelial coverage of the grafts of experimental animals. Herring et al., "A Single and Staged Technique for Seeding Vascular Grafts with Autogenous Endothelium", Surgery 1978;84:498-504; Graham et al., "Cultured Autogenous Endothelial Cell Seeding of Vascular Prosthetic Grafts", Surg Forum 1979;30:204-6; Graham et al., "Expanded Polytetrafluoroethylene Vascular Prostheses Seeded with Enzymatically Derived and Cultured Canine Endothelial Cells", Surgery 1982;91:550-9. Once covered by endothelium, grafts in dogs have been shown to be less thrombogenic as measured by platelet re-activity, to be more resistant to inoculation from blood-borne bacterial challenge, and to have prolonged patency of small-caliber vascular grafts. For a thorough listing of such experiments, see U.S. Pat. No. 5,131,907, supra.
A point of major concern when translating to human graft seeding has been the ability to produce enough endothelial cells with the use of human vascular tissue to allow seeding at a density high enough to attain endothelial cell coverage of the graft. Watkins et al., using human saphenous vein remnants following coronary artery bypass surgery, were able to produce small quantities of endothelial cells in culture and report a low-fold increase in confluent cell area obtained in culture after 4-6 weeks. Watkins et al., "Adult Human Saphenous Vein Endothelial Cells: Assessment of Their Reproductive Capacity for Use in Endothelial Seeding of Vascular Prostheses", J Surg Res 1984;36:588-96.
Even if it were possible to substantially expand the number of endothelial cells available through vigorous culturing techniques, concerns would still remain concerning the "health" of these endothelial cells after as many as 40 or 50 population doublings. Furthermore, the incubation of such cells in cultures that are foreign to their natural environment raises further concerns about genetic alterations and/or patient contamination with viruses, toxins, or other damaging materials.
Many endothelialization procedures are suggested in the literature. Investigations in this area have been complicated by the diverse nature of the endothelium itself and by the species to species differences that have been found relating to the behavior and characteristics of the endothelium. Fishman, "Endothelium A Distributed Organ of Diverse Capabilities", Annl of NY Acad of Sci 1982:1-8; Sauvage et al., "Interspecies Healing of Porous Arterial Prostheses", Arch Surg 1974;109:698-705; and Berger, "Healing of Arterial Prostheses in Man: Its Incompleteness", supra. Nonetheless, the literature is replete with reports of experiments involving the seeding of endothelial cells on various grafts, in various species, with a mixture of results. For a thorough listing of such experiments, see U.S. Pat. No. 5,131,907, supra.
It has been previously recognized that human microvascular endothelial cells, that is, the cells which are derived from capillaries, arterioles, and venules, will function suitably in place of large vessel cells even though there are morphological and functional differences between large vessel cells and microvascular endothelial cells in their native tissues.
U.S. Pat. No. 5,131,907, supra, describes the treatment to confluence of a vascular graft or other implant using microvascular endothelial cells that are separated from fat that is obtained at the beginning of an uninterrupted surgical procedure: in brief, fat tissue is removed from the patient after sterile conditions have been established, microvascular endothelial cells in that fat are then quickly separated from their related tissue by enzymatic digestion and centrifugation, and the cells are deposited on a surface by gravity or by filtration, which surface is then implanted into the patient during the latter stages of the same operation. A second uninterrupted surgical procedure for the creation of such vascular grafts that utilizes a crude fat slurry is also described: in brief, fat tissue is removed from the patient after sterile conditions have been established, the fat is homogenized to form a cellular slurry, this slurry is applied to an implant, and then this implant is implanted into the patient during the latter stages of the same operation. The synthetic vascular graft created by this protocol possesses a seeded monolayer of endothelial cells that is sufficient to promote spontaneous confluent endothelialization in vivo after implantation. This endothelialized graft is sufficiently thromboresistant to inhibit thrombosis, and, therefore, the use of this type of endothelialized graft substantially increases both graft patency and the surgical success rate of vascular grafting.
Additionally, U.S. Pat. No. 5,628,781, which is entitled "Improved Implant Materials, Methods of Treating the Surface of Implants with Microvascular Endothelial Cells, and the Treated Implants Themselves," describes a method for the creation of improved endothelialized vascular implants possessing substantially-enhanced anti-thrombogenic properties. First, the method describes the creation of a synthetic vascular graft possessing a monolayer of endothelial cells genetically modified to express any of a wide range of therapeutic agents. This enhanced expression of therapeutic agents, comprising the anti-thrombogenic proteins described hereinabove, would substantially improve graft patency and reduce localized thrombosis. Second, this method describes the use of improved polymers for the enhancement of graft anti-thrombosis. It has been observed that endothelial cells exhibit reduced thrombogenicity when in contact with different matrix proteins of the basement membrane as compared to tissue collagen. Implant materials can be treated by glow-discharge plasma modification to produce a surface rich in amines that possesses properties similar to those of the basement membrane. Thus this modified material induces these improved anti-thrombotic properties of endothelial cells normally stimulated by the basement membrane.
Additionally, some of the present inventors have engaged in substantial prior research work relating to the field of this invention, with numerous scientific publications and U.S. and foreign patents. The U.S. patents included in this work comprise: U.S. Pat. No. 4,820,626, U.S. Pat. No. 4,883,755, U.S. Pat. No. 5,035,708, U.S. Pat. No. 5,194,373, U.S. Pat. No. 5,230,693, U.S. Pat. No. 5,312,380, U.S. Pat. No. 5,372,945, and U.S. Pat. No. 5,441,539. The scientific publications included in this work comprise: Williams, "Endothelial Cell Transplantation", Cell Trans. 1995;4:401-409; and Wilson et al., "Implantation of Vascular Grafts Lined with Genetically Modified Endothelial Cells", Science 1989;244:1344-1346. These publications and patents provide further insight into and background for the present invention as well as additional methodological direction and description, and, thus, these publications and patents are incorporated by reference herein in their entirety.
In an improvement to this technique of Williams et al., U.S. Pat. No. 5,336,615 describes the genetic modification of endothelial cells with an additional gene, specifically the c-src gene, which increases the migrative ability of endothelial cells, and the subsequent vascular transplantation of these modified cells, whether directly or as a monolayer coating on a synthetic vascular graft, in order to increase endothelial cell migration onto the graft and to decrease thrombus formation. This patent provides an additional overview of many of the background procedures relating to the present invention and is therefore incorporated herein in its entirety.
Intimal Thickening
The second principal cause of vascular graft failure is the development of vascular stenosis, the narrowing of the arterial or venous lumen, due to expansive growth of the innermost cellular layer of a blood vessel, such growth known in the art as intimal thickening (generic) or intimal hyperplasia, in response vascular damage. As part of the inflammatory and reparative response to vascular damage, such as that resultant from vascular surgeries, inflammatory cells, including monocytes, macrophages, and activated polymorphonuclear leukocytes and lymphocytes, often form inflammatory lesions in the blood vessel wall. This formation induces activation of cells in the intimal and medial cellular layers of the blood vessel or heart. This activation may include the migration of cells to the innermost cellular layers, known as the intima. Such migrations pose a problem for the long-term success of vascular grafts because endothelial cells release smooth muscle cell growth factors, such as platelet-derived growth factor, interleukin-1, tumor necrosis factor, transforming growth factor-beta, and basic fibroblast growth factor, that cause these newly-migrated smooth muscle cells to proliferate. Additionally, thrombin has been demonstrated to promote smooth muscle cell proliferation both by acting as a growth factor itself and by enhancing the release of several other growth factors produced by platelets and endothelial cells. Wu et al., "Role of Endothelium in Thrombosis and Hemostasis", Annu Rev Med 1996;47:315-31. This proliferation causes irregular and uncontrolled growth of the intima into the lumen of the blood vessel or heart, which constricts and often closes the vascular passage. As utilized hereinafter, the term "intimal hyperplasia" is meant to refer specifically to the proliferation of smooth muscle cells present in the intima. Often, irregular calcium deposits in the media or lipid deposits in the intima accompany these growths, such lipid deposits normally existing in the form of cholesterol and cholestryl esters accumulated within macrophages, T lymphocytes, and smooth muscle cells, and these calcium and lipid deposits cause arteriosclerotic hardening of the arteries and veins and eventual vascular failure. These arteriosclerotic lesions caused by vascular grafting can also be removed by additional reconstructive vascular surgery, but the failure rate of this approach due to restenosis has been observed to be between thirty and fifty percent.
Because such surgical therapies have proven unsuccessful, many pharmacological treatments have been proposed for the treatment of vascular stenosis, though these treatments have met with limited success. One such proposed treatment has focused on the use of the secretory T lymphocyte protein interferon-gamma (.gamma.-IFN), which has been demonstrated to be a potent inhibitor of smooth muscle cell proliferation. The isolation and characterization of .gamma.-IFN is described in detail in U.S. Pat. No. 5,096,705, which is incorporated by reference herein in its entirety. Parenteral administration of .gamma.-IFN has been suggested as a potential treatment for vascular stenosis. Recombinant .gamma.-IFN was demonstrated by Hansson et al. to inhibit the proliferation of exponentially replicating smooth muscle cells in vitro, and a dose-dependent relationship was found to exist between .gamma.-IFN dose and inhibition of cell proliferation. Hansson et al., "Interferon-.gamma. Regulates Vascular Smooth Muscle Cell Proliferation and Ia Expression In Vivo and In Vitro", Circ Res 1988;63:712-719. Additional evidence obtained in this investigation suggested that .gamma.-IFN acts by blocking the transition from G.sub.0 to G.sub.1 or an early event during the G.sub.1 phase of the cell cycle in vascular smooth muscle cells. It was also observed that low levels of .gamma.-IFN are secreted locally within the intima by activated T lymphocytes during the normal vascular response to injury, and thus the production of .gamma.-IFN may be a part of the natural cellular immune and reparative response to vascular lesions. Hansson et al., supra. In subsequent work, Hansson et al. have demonstrated that parenteral administration of recombinant .gamma.-IFN in a murine model reduced the size of intimal lesions by as much as fifty percent. Hansson et al., "Interferon-.gamma. Inhibits Arterial Stenosis After Injury", Circ 1991;84:1266-1272. In this study, a marked reduction in the growth rate of smooth muscle cells within vascular lesions was only observed during the first two weeks of .gamma.-IFN administration, suggesting that the ultimate reduction in vascular lesion size may be due to this initial inhibition of cellular proliferation. However, complete inhibition of intimal hyperplasia and subsequent vascular failure was not achieved. Hansson et al. have received a U.S. Pat., No. 5,208,019, relating to this work. Thus, though administration of recombinant .gamma.-IFN has significant potential as a treatment for smooth muscle cell proliferation during vascular stenosis, no currently viable method for that treatment exists. Other pharmacological therapies, such as the administration of heparin, calcium channel blockers, and angiotensin antagonists, have been proposed and tested, and these therapies have also proven inadequate to inhibit intimal hyperplasia secondary to vascular surgery.
Because these surgical and pharmacological therapies for the control of smooth muscle cell proliferation and intimal hyperplasia have to date been unsuccessful in human clinical trials, genetic modification of endothelial cells has been proposed as a novel method for the targeted inhibition of vascular stenosis and treatment of arteriosclerosis. Hansson et al. and Wu et al., supra. Gene therapy offers several advantages in the inhibition of smooth muscle cell growth and intimal hyperplasia by directly affecting the microenvironment of the blood vessel. Recombinant gene expression may provide a more-nearly physiological production of target factors and would alleviate the need for repeated infusions of large quantities of an exogenous preparation of such factors, which could cause considerable side effects. A number of transformation vectors and protocols have been predicted to be potentially useful in the transfer of gene constructs into endothelial cells, including, but not limited to, the following: (i) viral vectors, such as adenovirus, retroviruses, and adeno-associated viruses, (ii) non-viral vectors, such as cationic lipids and targeted polylysine-DNA condensation, (iii) electrophoretic methods, (iv) calcium-phosphate techniques, and (v) metaloprojectile transformation methods utilizing tungsten, gold, or other such suitable metals.
Other vectors having characteristics useful in the transformation of endothelial cells will be apparent to those skilled in the art. The term "vector" is well understood in the art and refers to any vehicle for the transformation of a cell or organism. Additionally, the term "transformation" is well understood in the art and refers to the addition of genetic material into a cell or organism, such "genetic material" understood by those skilled in the art to consist of "genes" or parts thereof, which are DNA or RNA sequences, whether synthetic or naturally occurring, that encode a functional protein or RNA molecule. Proposals for the use of gene therapy normally involve the transformation of endothelial cells in vivo with adenovirus and lipofectin-Sendai viruses and have been successfully accomplished for the transformation of prostaglandin H synthase-1 and nitric oxide synthase-III (NOS-III). Wu et al., "Restoration of Prostacyclin Synthesis by Transfer of PGHS cDNA", Adv Prostagl Thrombox Leukotri 1994;23:377-80 and Von der Leyen et al., "Gene Therapy Inhibiting Neointimal Vascular Lesion: In Vivo Transfer of Endothelial Cell Nitric Oxide Synthase Gene", Proc Natl Acad Sci USA 1995;92:1137-47. The NOS-III transformation study by Von Der Leyen et al. utilizing a murine model is the only study to date that has documented any therapeutic effects with in vivo gene transfer, though the in vivo method presented in this study was unable to completely inhibit intimal hyperplasia. Additionally, in vivo protocols for the treatment of pre-existing arteriosclerotic lesions would be further hampered by the fact that viral transformation efficiency is markedly reduced when transforming arteriosclerotic blood vessels as compared to those without lesions. Feldman et al., "Low Efficiency of Percutaneous Adenovirus-Mediated Arterial Gene Transfer in the Atherosclerotic Rabbit", J Clin Invest 1995,95:2662-71. This is a significant problem for in vivo methodologies, because high titers of these vectors have been demonstrated to elicit immunologic reactions and vascular inflammatations. Simon et al., "Adenovirus-Mediated Gene Transfer of the CFTR Gene to Lungs of Nonhuman Primates: Toxicity Study", Hum Gene Ther 1993;4:771-80. With the existence of these significant limitations, such in vivo methodologies for the treatment of vascular stenosis are currently unfeasible.
Thus, there exists a need for a medical treatment for vascular, heart, and renal failures that inhibits both thrombogenicity and smooth muscle cell proliferation secondary to surgery.